Studies from the laboratory of the PI have focused on elucidating the components, mechanisms, and roles of bioactive sphingolipids, especially ceramide. These have resulted in identification of stress (TNF, heat, chemotherapy agents)- induced activation of neutral sphingomyelinase (SMase). Recent advances have included identification of the orthologue of SMase in S. cerevisiae, ISC1 which acts on membrane inositolphosphoceramide (IPC) to liberate ceramide. This enzyme shows homology to a mammalian enzyme, nSMase2, which is poorly characterized. We propose the overall hypothesis that these enzymes define a family of sphingolipid phosphodiesterases with similar mechanisms of action in vitro and with roles in regulating the levels of SM/1PC/ceramide in yeast and mammals. This hypothesis has generated the following specific aims: (1) To determine the physiologic function of nSMase 2. This aim will test the specific hypothesis that nSMase2 functions as an endogenous SMase. This will be determined through a detailed investigation of the effects of this enzyme on basal and agonist-stimulated sphingolipid metabolism. (2) To determine in vitro mechanisms of ISC1 and nSMase 2. This aim will adopt a combination of molecular and biochemical approaches to determine specific domains in the enzymes that interact with substrate, the specific determinants of substrate selectivity, and domains responsible for regulation by phosphatidylserine. (3) To determine cellular roles of ISC1, This aim will test the spedfie hypothesis that ISC1 launches a sphingolipid-mediated pathway that functions in response to heat stress in yeast. This will be investigated by employing a combination of microarray expression, biochemical studies, and molecular genetic approaches to define specific and general pathways regulated by ISC1 and its product ceramide. Understanding the mechanisms, regulation, and function of these enzymes is critical for the study of bioactive lipids in general and sphingolipids in particular. These novel and important enzymes will therefore open up a novel area of research, hitherto resistant to molecular approaches. These pathways are of direct significance to the biology of stress responses and specifically to cancer therapeutics.